The N-acetylglucosamine (O-GlcNAc) modification is a reversible attachment of O-GlcNAc onto serine or threonine residues of intracellular proteins. This modification mediates cellular activities by regulating protein trafficking, conformational change, and by antagonizing phosphorylation. Many human pathologies exhibit aberrant O-GlcNAcylation of specific proteins. Current strategies for detecting O-GlcNAc, however, are insufficient. Immunoblotting and mass spectrometry are the state-of-the-art methods to study protein O-GlcNAcylation. To detect the modification of a specific protein, Western blots are typically paired with a prior precipitation step with either target specific antibodies or anti-O-GlcNAc affinity reagents. This enrichment process can be labor intensive and lead to major sample loss due to the relatively low affinity of lectins and anti-O-GlcNAc antibodies. Mass spectrometry is the most widely used tool for profiling protein-specific O-GlcNAc modification on a global level. The generation of high-quality glycoproteomics data, however, requires large quantities of sample followed by rigorous and labor intensive enrichment to ensure adequate representation of low-abundance glycoproteins. Also, the instrumentation to perform these analyses is possessed by only a few labs due to it being very expensive and requiring a high level of specialized expertise to operate. We herein propose to develop a low-tech, broadly accessible method for analyzing the O-GlcNAcylation state of specific proteins. Changes in O-GlcNAcylation are mechanistically implicated in many human diseases and thus represent a fertile ground for scientific investigation. The broad goal of this proposal is to develop and disseminate an accessible PCR-based glycoproteomics platform for monitoring changes in O-GlcNAcylation. We describe the development of a technique that will allow the detection of protein-specific glycosylation directly from lysate using chemical probes of O-GlcNAc in tandem with multiplexed proximity ligation assays and fluorescence-based quantitative PCR.